Human cytomegalovirus (HCMV) causes one of the most common opportunistic infections in patients with AIDS. Disseminated HCMV infection in these patients is usually associated with gastroenteritis, pneumonia, and sight-threatening retinitis. The emergence of drug-resistant HCMV strains to currently available drugs (e.g. ganciclovir) has posed a need to develop new drugs and novel strategies to combat HCMV infections. The objective of this study is to develop Salmonella as a novel class of gene transfer vectors for targeted delivery of anti-HCMV RNase P ribozymes for blocking viral infection and preventing viral diseases. Attenuated strains of Salmonella can function as a carrier system for the delivery of eukaryotic expression vectors in vitro and in vivo and have been shown to deliver therapeutic agents, including nucleic acids-based vaccines and anti-tumor molecules (e.g. small interfering RNAs) for the treatment and prevention of human diseases. We have recently shown that attenuated Salmonella can efficiently deliver an anti-HCMV RNase P ribozyme sequence in human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. However, whether novel Salmonella strains can be constructed for efficient delivery of RNase P ribozymes in cells, and whether Salmonella-mediated delivery of RNase P ribozymes is specific and effective in inhibiting viral pathogenesis in animal models have not been shown. Equally elusive is the mechanism of how Salmonella achieves efficient gene transfer for delivery of nucleic acids-based agents (e.g. RNase P ribozymes) in human cells. To address these issues, we will first propose to generate novel attenuated Salmonella strains that efficiently carry out gene transfer for RNase P ribozyme expression in human cells and study the mechanisms of the gene transfer. We will then investigate whether targeted delivery of RNase P ribozyme by the newly generated Salmonella abolishes HCMV gene expression and growth in cultured human cells. Finally, using murine cytomegalovirus (MCMV) infection of mice as a model system, we will determine whether the generated Salmonella strains are highly efficient for targeted delivery of RNase P ribozyme in animals and whether the targeted delivery of RNase P ribozymes mediated by Salmonella is highly effective in blocking MCMV infection and pathogenesis in vivo. Our research will generate novel Salmonella strains with efficient gene transfer activity, and will provide insight into how Salmonella achieves efficient gene delivery in mammalian cells. We will also determine whether Salmonella-mediated gene delivery of RNase P ribozymes is specific and effective in shutting down gene expression in cultured cells and in animals. This study will facilitate the development of Salmonella-mediated gene transfer of RNase P ribozymes as a novel therapeutic approach for treatment of infections by HCMV and other AIDS-associated viruses. PUBLIC HEALTH RELEVANCE: The proposed research is to generate novel agents for targeted gene delivery of antiviral molecules in treating infections caused by AIDS-associated viruses, including human cytomegalovirus (HCMV), which causes one of the most common opportunistic infections in AIDS patients. Our study will facilitate the development of a new method that can be used as a research tool and a therapeutic approach for studies and treatment of infections associated with human viruses such as HCMV.